KLF5 inhibits the migration and invasion in cervical cancer cell lines by regulating SNAI1.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is an important biological process by which malignant tumor cells to acquire migration and invasion abilities. This study explored the role of KLF5 in the EMT process of in cervical cancer cell lines. OBJECTIVE: Krüpple-like factor 5 (KLF5) is a basic transcriptional factor that plays a key role in cell-cycle arrest and inhibition of apoptosis. However, the molecular mechanism by which KLF5 mediates the biological functions of cervical cancer cell lines has not been elucidated. Here, we focus on the potential function of ELF5 in regulating the EMT process in in vitro model of cervical cancer cell lines. METHOD: Western-blot and real-time quantitative PCR were used to detect the expression of EMT-related genes in HeLa cells. MTT assays, cell scratch and Transwell assays were used to assess HeLa cells proliferation and invasion capability. Using the bioinformatics tool JASPAR, we identified a high-scoring KLF5-like binding sequence in the SNAI1 gene promoter. Luciferase reporter assays was used to detect transcriptional activity for different SNAI1 promoter truncates. RESULT: After overexpressing the KLF5 gene in HeLa cells, KLF5 not only significantly inhibited the invasion and migration of HeLa cells, but also increased the expression of E-cadherin and decreased the expression of N-cadherin and MMP9. In addition, the mRNA expression of upstream regulators of E-cadherin, such as SNAI1, SLUG, ZEB1/2 and TWIST1 was also decreased. Furthermore, KLF5 inhibiting the expression of the SNAI1 gene via binding its promoter region, and the EMT of Hela cells was promoted after overexpression of the SNAI1 gene. CONCLUSION: These results indicate that KLF5 can downregulate the EMT process of HeLa cells by decreasing the expression of the SNAI1 gene, thereby inhibiting the migration and invasion of HeLa cervical cancer cells.

Effects of ligustilide on chemotherapy resistance of cervical cancer cells / 中国药房

OBJECTIVE To investigate the effects of ligustilide on chemotherapy resistance of cervical cancer cells based on Hippo-Yes-associated protein （YAP） signaling pathway. METHODS Human cervical cancer cisplatin-resistant cells HeLa/DDP were divided into control group， cisplatin group （10 μmol/L cisplatin）， cisplatin+ligustilide low-， medium- and high-concentration groups （10 μmol/L cisplatin+25， 50， 100 μmol/L ligustilide）. The proliferation， apoptosis， migration and invasion of HeLa/DDP cells were all detected in each group. The mRNA expressions of YAP and transcriptional coactivator with PDZ binding motif （TAZ） as well as the protein expressions of YAP， TAZ， matrix metalloproteinase 2 （MMP2）， Ki67， cleaved-caspase-3 and caspase-3 were determined in HeLa/DDP cells. RESULTS Compared with control group， the inhibitory rate， apoptotic rate and cleaved- caspase-3/caspase-3 of cisplatin group were increased significantly； scratch healing rate， the number of invasive cells， the mRNA expressions of YAP and TAZ， and the protein expressions of YAP， TAZ， MMP2 and Ki67 were decreased significantly in cisplatin group （P＜0.05）. Compared with cisplatin group， the inhibitory rate of cell proliferation， apoptotic rate and cleaved-caspase-3/ caspase-3 were further increased in cisplatin+ligustilide low-， medium- and high-concentration groups， while scratch healing rate， the number of invasive cells， the mRNA expressions of YAP and TAZ， and the protein expressions of YAP， TAZ， MMP2 and Ki67 were further decreased， in a dose-dependent manner （P＜0.05）. CONCLUSIONS Ligustilide can increase the sensitivity of drug-resistant cervical cancer cells to cisplatin by inhibiting Hippo-YAP signaling pathway.

Mechanism of malignant behavior in cervical cancer cells based on NOD2-mediated AMPK/mTOR signaling pathway / 安徽医科大学学报

Objective To explore the mechanism of malignant behavior of cervical cancer(CC)cells based on AMP-activated protein kinase(AMPK)/rapamycin target protein(mTOR)signaling pathway mediated by nucleo-tide-binding oligomerization domain receptor 2(NOD2).Methods Bioinformatics analysis was performed to deter-mine the expression of NOD2 in CC tissue.Plasmids targeting NOD2(shNOD2)and shRNAs negative control(shNC),NOD2 overexpression(NOD2)and vectors(Vec)were transfected into CC cells.The effect of NOD2 on the growth of CC cells was determined by cell counting kit-8 assay,colony formation and Transwell cell invasion as-say.Transcriptome analysis was performed by high throughput RNA sequencing(RNA-Seq).Western blot was used to detect the expression of NOD2,AMPK/mTOR signaling pathway and autophagy protein in the cell line.24 female BALB/c nude mice were randomly divided into four groups,with 6 mice in each group:vector group(Vec group),NOD2 overexpression group(NOD2 group),shNC group and shNOD2 group.The distant metastasis mod-el was established in mice,and the fluorescence intensity of lung metastasis was monitored and the number of lung metastasis nodules was counted.Results On-line database analysis showed that the expression of NOD2 in CC tis-sues was significantly higher than that in normal tissues,and there were significant differences in the mRNA expres-sion of NOD2 in different stages of CC(P＜0.05).In addition,the high expression of NOD2 was associated with poor overall survival and disease-free survival(P＜0.05).NOD2 overexpression promoted the proliferation,colony formation,migration and invasion of CC cells,while NOD2 knock-down was the opposite.Consistent with the re-sults in vitro,the lung colonization and lung metastasis of CC cells in NOD2 group were significantly higher than those in Vec group(P＜0.05),while those in shNOD2 group were significantly lower than those in shNC group(P＜0.05).RNA-Seq results showed that the expression of NOD2 was significantly related to AMPK signal activa-tion,mTOR signal inhibition,autophagy regulation pathway activation and autophagy formation.Compared with shNC group,the expression of phosphorylated AMPK and LC3 protein decreased significantly in shNOD2 group(P＜0.05),and the expression levels of phosphorylated mTOR and p62 protein increased significantly(P＜0.05).Compared with Vec group,the expression levels of LC3 and AMPK protein in NOD2 group increased significantly(P＜0.05),and the expression levels of phosphorylated mTOR and p62 protein decreased significantly(P＜0.05).Compared with shNC group,the point accumulation of GFP-mRFP-LC3 in shNOD2 group decreased signifi-cantly(P＜0.05).Compared with Vec group,the point accumulation of GFP-mRFP-LC3 increased significantly(P＜0.05).Conclusion NOD2 may promote the proliferation,migration and invasion of CC through AMPK/mTOR signal,and its mechanism partly involves autophagy activation.

CircRNA_PLEKHM3 regulates epithelial mesenchymal transformation of cervical cancer cells through the miR-320/KLF4 axis / 安徽医科大学学报

Objective @#To investigate the role and mechanism of circular RNA containing pleck strin homology do main of Pleck strin homology domain family M member 3 ( circRNA_PLEKHM3) in regulating epithelial mesenchy mal transition (EMT) behavior in cervical cancer cells through the miR-320 and KLF4 .@*Methods @#The expression levels of circRNA_PLEKHM3 in cervical cancer cells Hela and CaSki were detected by real time quantitative PCR (qRT PCR) . RNA fluorescence in situ hybridization was used to determine the localization of circRNA_PLEKHM3 in human cervical cancer epithelial cells CaSki . Dual luciferase reporter gene experiments were conducted to inves tigate the targeting relationship between circRNA_PLEKHM3 and miR-320 , as well as the targeting relationship be tween miR-320 and KLF4 . CaSki cells were overexpressed with circRNA_PLEKHM3. Additionally, three groups were set up: overexpression of miR-320 on the basis of circRNA_PLEKHM3 overexpression , silencing of KLF4 on the basis of circRNA_PLEKHM3 overexpression , and silencing of KLF4 on the basis of miR-320 overexpression . qRT PCR was performed to detect the expression levels of miR-320 in CaSki . Western blot experiments were con ducted to determine the expression of KLF4 and EMT markers including E-cadherin , N-cadherin , Vimentin , MMP-2 , and MMP-9 in CaSki cells . Transwell assays were performed to measure cell migration and invasion .@*Results@#The expression of circRNA_PLEKHM3 decreased in Hela and CaSki cells (P < 0 05) , mainly localized in the cytoplasm . The dual luciferase reporter gene experiment demonstrated a targeting relationship between miR-320 and circRNA_PLEKHM3 , as well as between KLF4 and miR-320 . Overexpression of circRNA_PLEKHM3 inhibited the protein expression of miR-320 , Ncadherin , Vimentin , MMP-2 , and MMP-9 , up regulated the protein expression of E-cadherin, and reduced cell migration and invasion ( P < 0.05) . Overexpression of miR 320 or silencing of KLF4 on the basis of circRNA_PLEKHM3 overexpression both promoted the protein expression of miR-320, N-cad herin , Vimentin , MMP-2 , and MMP-9 , down regulated the protein expression of E-cadherin , and increased cell migration and invasion (P < 0.05) . However , silencing of KLF4 on the basis of miR-320 overexpression inhibited the protein expression of KLF4 , N-cadherin , Vimentin , MMP-2 , and MMP-9 , up regulated the protein expression of E-cadherin , and reduced cell migration and invasion ( P < 0.05) . @*Conclusion @#Overexpression of circRNA _PLEKHM3 regulates EMT in cervical cancer cells through the miR -320/KLF4 axis .

Phytochemical profile and biological activities of Bromelia antiacantha extracts / Perfil fitoquímico e atividades biológicas de extratos de Bromelia antiacantha

Reports from popular medicine usually act as a basis for the development of new drugs from natural compounds with therapeutic actions for serious diseases and prevalence such as cancer. Bromelia antiacantha Bertol. is a species of the Bromeliaceae family, considered an unconventional food plant, found in the south and midwest regions of Brazil. Despite the high nutritional content and pharmacological potential of its fruits, few scientific studies report its biological actions. Thus, this study evaluates the phytochemical profile of aqueous and ethanol extracts obtained from B. antiacantha fruits, as well as their possible antioxidant, antitumor, and cytotoxic activities. The aqueous extract exhibited phenolic compounds and flavonoids, while ethanol extracts indicated the presence of flavonoids and coumarin in their composition, regardless of the region of collection. The ethanolic extract demonstrated a more promising antioxidant effect than the aqueous extract and also induced a significant inhibition in the viability of human cervical cancer cells of the SiHa strain. In addition, treatment with both extracts did not alter the viability of non-tumor cells of the immortalized human keratinocyte lineage (HaCaT). These results bring new data about extracts obtained from a native plant, edible and traditionally used in popular medicine, opening new perspectives for its possible therapeutic application.

Relatos da medicina popular costumam atuar como referencial para o desenvolvimento de novos fármacos a partir de moléculas naturais com ações terapêuticas para doenças de alta gravidade e prevalência como o câncer. Bromelia antiacantha Bertol. é uma espécie da família Bromeliaceae, considerada uma planta alimentícia não convencional (PANC), encontrada nas regiões sul e centro-oeste do Brasil. Apesar do alto teor nutritivo e potencial farmacológico de seus frutos, poucos estudos científicos relatam suas ações biológicas. Desta forma, este estudo avalia o perfil fitoquímico de extratos aquoso e etanólico obtidos de frutos de B. antiacantha, bem como a sua possível ação antioxidante, antitumoral e citotóxica. O extrato aquoso apresentou compostos fenólicos e flavonoides, enquanto os extratos etanólicos apontam a presença de flavonóides e cumarina em sua composição, independente da região de coleta. O extrato etanólico demonstrou efeito antioxidante mais promissor do que o extrato aquoso e também induziu uma inibição significativa na viabilidade de células humanas de câncer cervical da linhagem SiHa. Além disso, o tratamento com ambos extratos não alterou a viabilidade de células não tumorais da linhagem de queratinócitos humanos imortalizados (HaCaT). Estes dados trazem novas informações sobre extratos obtidos de uma espécie vegetal nativa, comestível e já utilizada tradicionalmente, mas abrindo novas perspectivas quanto a possíveis aplicações terapêuticas.

Phytochemical profile and biological activities of Bromelia antiacantha extracts

Abstract Reports from popular medicine usually act as a basis for the development of new drugs from natural compounds with therapeutic actions for serious diseases and prevalence such as cancer. Bromelia antiacantha Bertol. is a species of the Bromeliaceae family, considered an unconventional food plant, found in the south and midwest regions of Brazil. Despite the high nutritional content and pharmacological potential of its fruits, few scientific studies report its biological actions. Thus, this study evaluates the phytochemical profile of aqueous and ethanol extracts obtained from B. antiacantha fruits, as well as their possible antioxidant, antitumor, and cytotoxic activities. The aqueous extract exhibited phenolic compounds and flavonoids, while ethanol extracts indicated the presence of flavonoids and coumarin in their composition, regardless of the region of collection. The ethanolic extract demonstrated a more promising antioxidant effect than the aqueous extract and also induced a significant inhibition in the viability of human cervical cancer cells of the SiHa strain. In addition, treatment with both extracts did not alter the viability of non-tumor cells of the immortalized human keratinocyte lineage (HaCaT). These results bring new data about extracts obtained from a native plant, edible and traditionally used in popular medicine, opening new perspectives for its possible therapeutic application.

Resumo Relatos da medicina popular costumam atuar como referencial para o desenvolvimento de novos fármacos a partir de moléculas naturais com ações terapêuticas para doenças de alta gravidade e prevalência como o câncer. Bromelia antiacantha Bertol. é uma espécie da família Bromeliaceae, considerada uma planta alimentícia não convencional (PANC), encontrada nas regiões sul e centro-oeste do Brasil. Apesar do alto teor nutritivo e potencial farmacológico de seus frutos, poucos estudos científicos relatam suas ações biológicas. Desta forma, este estudo avalia o perfil fitoquímico de extratos aquoso e etanólico obtidos de frutos de B. antiacantha, bem como a sua possível ação antioxidante, antitumoral e citotóxica. O extrato aquoso apresentou compostos fenólicos e flavonoides, enquanto os extratos etanólicos apontam a presença de flavonóides e cumarina em sua composição, independente da região de coleta. O extrato etanólico demonstrou efeito antioxidante mais promissor do que o extrato aquoso e também induziu uma inibição significativa na viabilidade de células humanas de câncer cervical da linhagem SiHa. Além disso, o tratamento com ambos extratos não alterou a viabilidade de células não tumorais da linhagem de queratinócitos humanos imortalizados (HaCaT). Estes dados trazem novas informações sobre extratos obtidos de uma espécie vegetal nativa, comestível e já utilizada tradicionalmente, mas abrindo novas perspectivas quanto a possíveis aplicações terapêuticas.

circRNA THBS1 silencing inhibits the malignant biological behavior of cervical cancer cells via the regulation of miR-543/HMGB2 axis.

Circular RNA (circRNA) THBS1 has been shown to exist as an oncogene in non-small-cell lung cancer, but its role in cervical cancer is still unclear. Our experiment aimed to uncover the functions and specific mechanism of circRNA THBS1 in cervical cancer cells. Levels of circRNA THBS1 and miR-543 in cervical cancer tissues and cell lines were assessed by RT-qPCR. starBase and dual luciferase reporter gene assay were applied for investigating the correlation between miR-543 and circRNA THBS1/HMGB2. Cell proliferation and apoptosis were evaluated by MTT and flow cytometry, respectively. Furthermore, the levels of HMGB2, E-cadherin, and N-cadherin in HeLa cells were determined by RT-qPCR and western blot analysis. Our data revealed that circRNA THBS1 was significantly upregulated and miR-543 was low expressed in cervical cancer tissues and cell lines. circRNA THBS1 interacted with miR-543 and negatively regulated miR-543 expression in HeLa cells. Silencing of circRNA THBS1 remarkably suppressed HeLa cells' viability, accelerated cells' apoptosis, and inhibited the EMT of HeLa cells, while these changes were reversed by miR-543 inhibitor. Moreover, miR-543 affected HeLa cells by targeting HMGB2. In conclusion, circRNA THBS1 silencing inhibited the malignant biological behaviors of cervical cancer cells via the regulation of miR-543/HMGB2 axis.

Nanoscale insight into biochemical changes in cervical cancer cells exposed to adaptogenic drug.

This paper describes for the first time the application of atomic force microscopy-based infrared spectroscopy (AFM-IR) to evaluate cellular response to adaptogen, based on an in vitro model of cervical cancer. HeLa cervical cells were exposed to different concentrations of withaferin A, a very promising anti-cancer adaptogenic substance. AFM-IR approach was used to image single cells post-adaptogen treatment and to track subtle biochemical changes in cells at the nanoscale level. Partial least squares (PLS) regression was applied to build predictive models that allowed for the identification of spectral markers of adaptogen-induced alterations Spectroscopic studies were enriched with fluorescence staining to determine whether the adaptogen affects cell morphology. The results showed that with the increase in the concentration of adaptogen, changes in the cell nucleus and the actin cytoskeleton become more and more significant. It has been demonstrated that the AFM-IR technique can successfully study the cellular response to the anti-cancer agent at the single-cell level with nanoscale spatial resolution. On the basis of the promising findings presented in this paper, it is possible to conclude that withaferin A has great potential in inhibiting the proliferation of cervical cancer cells in a dose-dependent manner. It has been found that both the increase in the concentration of withaferin A and the increase in incubation time with the adaptogen resulted in a decrease in the intensity of the bands assigned to nucleic acids. This may be due to DNA condensation, internuclear cleavage, or degradation during apoptosis. The findings also suggest changes in the secondary structure of proteins that may be a consequence of disruption of the actin cytoskeleton, progressive apoptosis, or significant biochemical changes. Furthermore, noticeable changes were also observed in the bands originating from lipids vibrations, and an increased share of the band near 2920 cm-1, considered an important marker of apoptosis, was noted. The metabolism of carbohydrates in cells also changes under the influence of the adaptogen. AFM-IR provides nanoscale insight into the structural and morphological properties of cells after drug treatment and is an indisputable milestone in the development of new anti-cancer approaches.

Fu Fang Gang Liu aqueous extract inhibits the proliferation of HeLa cells by causing deoxyribonucleic acid damage.

ETHNOPHARMACOLOGICAL RELEVANCE: Fu Fang Gang Liu (FFGL) is an effective formula for treating wart proliferation caused by human papillomavirus (HPV) infection and has the potential to treat HPV-related cancers. However, scientific evidence of its anti-tumor activity against cervical cancer, the most common cancer caused by HPV, is lacking. AIM OF THE STUDY: To clarify the anti-tumor effect of an FFGL aqueous extract on human cervical cancer and its possible mechanism of cell cycle arrest in HeLa cells. MATERIALS AND METHODS: The anti-proliferative effect of FFGL on cervical cancer cells was assessed using the cell counting kit-8 assay. The proportion of apoptotic cells, cell cycle distribution, and cell division rate were determined using flow cytometry. Quantitative proteomics was used to identify differentially expressed proteins after FFGL treatment, and bioinformatics analysis was used to identify key nodal proteins affected by FFGL. Immunofluorescence and western blot analyses were used to explore changes in the expression of related proteins in the cell cycle and DNA damage pathways to elucidate the potential mechanism of action of FFGL against HeLa cell proliferation. RESULTS: FFGL inhibited cervical cancer cell proliferation and caused cell cycle arrest. According to quantitative proteomics, CyclinB1 may play an important role in the anti-proliferative effect of FFGL on HeLa cells. Additional experiments showed that FFGL aqueous extract caused ATM-mediated DNA damage, further phosphorylated CHK2, led to the inactivation of Cdc25C, inhibited the activity of the CDK1/CyclinB1 complex, and resulted in cell cycle arrest. CONCLUSIONS: FFGL can inhibit cervical cancer cell proliferation. Furthermore, it can increase CDK1 phosphorylation, block the cell cycle by causing DNA damage, and inhibit HeLa cell proliferation.

Adaptogenic activity of withaferin A on human cervical carcinoma cells using high-definition vibrational spectroscopic imaging.

Despite invaluable advances in cervical cancer therapy, treatment regimens for recurrent or persistent cancers and low-toxicity alternative treatment options are scarce. In recent years, substances classified as adaptogens have been identified as promising drug sources for preventing and treating cancer-based diseases on their ability to attack multiple molecular targets. This paper establishes the effectiveness of inhibition of the neoplastic process by a withaferin A (WFA), an adaptogenic substance, based on an in vitro model of cervical cancer. This study explores for the first time the potential of high-definition vibrational spectroscopy methods, i.e. Fourier-transform infrared (FT-IR) and Raman spectroscopic (RS) imaging at the single-cell level to evaluate the efficacy of the adaptogenic drug. HeLa cervical cancer cells were incubated with various concentrations of WFA at different incubation times. The multimodal spectroscopic approach combined with partial least squares (PLS) regression allowed the identification of molecular changes (e.g., lipids, protein secondary structures, or nucleic acids) induced by WFA at the cellular level. The results clearly illustrate the enormous potential of WFA in inhibiting the proliferation of cervical cancer cells. WFA inhibited the growth of the studied cancer cell line in a dose-dependent manner. Such studies provide comprehensive information on the sensitivity of cells to adaptogenic drugs. This is a fundamental step towards determining the rate and nature of adaptogen-induced changes in cancer cells.

P2X7 receptor involved in antitumor activity of atractylenolide I in human cervical cancer cells.

Atractylenolide I (Atr-I) was found to sensitize a variety of human cancer cells in previous studies. Purinergic P2X7R plays important role in different cancers. However, whether Atr-I could generate antitumor activity in human cervical cancer cells and P2X7R get involved in this effect remain unclear. In this study, Hela (HPV 18 +) and SiHa (HPV 16 +) cells were treated with different doses of Atr-I. The results indicated that agonist and antagonist of P2X7 receptors, BzATP and JNJ-47965567 (JNJ), could suppress the proliferation of Hela and SiHa cells. Atr-I demonstrated a considerable antitumor effect in both human cervical cancer cells in vitro. Atr-I combined with P2X7R agonist, BzATP, restored Atr-I-induced growth inhibition in Hela cells but not in SiHa cells. However, the combinatorial treatment of P2X7R antagonist JNJ and Atr-I has an additive effect on cell growth inhibition in SiHa cells rather than in Hela cells. It implied that P2X7R would get involved in the anti-human cervical cancer cells effect of Atr-I.

Exogenous growth hormone promotes an epithelial-mesenchymal hybrid phenotype in cancerous HeLa cells but not in non-cancerous HEK293 cells.

In cancer, the Epithelial to Mesenchymal Transition (EMT) is the process in which epithelial cells acquire mesenchymal features that allow metastasis, and chemotherapy resistance. Growth hormone (GH) has been associated with melanoma, breast, and endometrial cancer progression through an autocrine regulation of EMT. Since exogenous and autocrine expression of GH is known to have different molecular effects, we investigated whether exogenous GH is capable of regulating the EMT of cancer cells. Furthermore, we investigated whether exogenous GH could promote EMT in non-cancerous cells. To study the effect of GH (100 ng/ml) on cancer and non-cancer cells, we used HeLa and HEK293 cell lines, respectively. We evaluated the loss of cell-cell contacts, by cell scattering assay and migration by wound-healing assay. Additionally, we evaluated the morphological changes by phalloidin-staining. Finally, we evaluated the molecular markers E-cadherin and vimentin by flow cytometry. GH enhances cell scattering and the migratory rate and promotes morphological changes such as cell area increase and actin cytoskeleton filaments formation on HeLa cell line. Moreover, we found that GH favors the expression of the mesenchymal protein vimentin, followed by an increase in E-cadherin's epithelial protein expression, characteristics of an epithelial-mesenchymal hybrid phenotype that is associated with metastasis. On HEK293cells, GH promotes morphological changes, including cell area increment and filopodia formation, but not affects scattering, migration, nor EMT markers expression. Our results suggest that exogenous GH might participate in cervical cancer progression favoring a hybrid EMT phenotype but not on non-cancerous HEK293 cells.

ZIP11 Regulates Nuclear Zinc Homeostasis in HeLa Cells and Is Required for Proliferation and Establishment of the Carcinogenic Phenotype.

Zinc (Zn) is an essential trace element that plays a key role in several biological processes, including transcription, signaling, and catalysis. A subcellular network of transporters ensures adequate distribution of Zn to facilitate homeostasis. Among these are a family of importers, the Zrt/Irt-like proteins (ZIP), which consists of 14 members (ZIP1-ZIP14) that mobilize Zn from the extracellular domain and organelles into the cytosol. Expression of these transporters varies among tissues and during developmental stages, and their distribution at various cellular locations is essential for defining the net cellular Zn transport. Normally, the ion is bound to proteins or sequestered in organelles and vesicles. However, though research has focused on Zn internalization in mammalian cells, little is known about Zn mobilization within organelles, including within the nuclei under both normal and pathological conditions. Analyses from stomach and colon tissues isolated from mouse suggested that ZIP11 is the only ZIP transporter localized to the nucleus of mammalian cells, yet no clear cellular role has been attributed to this protein. We hypothesized that ZIP11 is essential to maintaining nuclear Zn homeostasis in mammalian cells. To test this, we utilized HeLa cells, as research in humans correlated elevated expression of ZIP11 with poor prognosis in cervical cancer patients. We stably knocked down ZIP11 in HeLa cancer cells and investigated the effect of Zn dysregulation in vitro. Our data show that ZIP11 knockdown (KD) reduced HeLa cells proliferation due to nuclear accumulation of Zn. RNA-seq analyses revealed that genes related to angiogenesis, apoptosis, mRNA metabolism, and signaling pathways are dysregulated. Although the KD cells undergoing nuclear Zn stress can activate the homeostasis response by MTF1 and MT1, the RNA-seq analyses showed that only ZIP14 (an importer expressed on the plasma membrane and endocytic vesicles) is mildly induced, which may explain the sensitivity to elevated levels of extracellular Zn. Consequently, ZIP11 KD HeLa cells have impaired migration, invasive properties and decreased mitochondrial potential. Furthermore, KD of ZIP11 delayed cell cycle progression and rendered an enhanced senescent state in HeLa cells, pointing to a novel mechanism whereby maintenance of nuclear Zn homeostasis is essential for cancer progression.

Enhanced Anti-Cancer Effects of Conditioned Medium from Hypoxic Human Adult Dermal Fibroblasts on Cervical Cancer Cells.

Hypoxia regulates fibroblast function by changing intracellular signaling and secretion factors, that influence the states of nearby cells. In this work, we investigated how medium (CM) from human adult dermal fibroblasts (HDFs) cultured in normoxic and hypoxic conditions affected cervical cancer (HeLa) cells. The HeLa cells showed decreased cell viability, increased apoptosis, and cell cycle arrest in response to CM from hypoxic-cultured HDFs (H-CM) compared with CM from normoxic-cultured HDFs (N-CM). Among the proteins up-regulated (>2-fold) in H-CM compared with N-CM, lymphotoxin-beta receptor (LTBR) decreased the viability of HeLa cells. Among the intracellular proteins down-regulated (>2-fold) in HeLa cells treated with H-CM compared with N-CM, the most enriched biological process GO term and KEGG pathway were protein deubiquitination and hsa05166:HTLV-I infection, respectively. In the protein−protein interaction network of intracellular proteins with altered expression (>2-fold), 1 up-regulated (TNF) and 8 down-regulated (ESR1, MCL1, TBP, CD19, LCK, PCNA, CHEK1, and POLA1) hub proteins were defined. Among the down-regulated hub proteins, the most enriched biological process GO term and KEGG pathway were leading strand elongation and hsa05166:HTLV-I infection, respectively. This study reveals that H-CM had stronger anti-cancer effects on cervical cancer cells than N-CM and induced intracellular signaling patterns related to those enhanced anti-cancer effects.

Inhibition of DDX3 and COX-2 by forskolin and evaluation of anti-proliferative, pro-apoptotic effects on cervical cancer cells: molecular modelling and in vitro approaches.

Several studies have reported up-regulation of both cyclooxygenase-2 (COX-2) and DEAD-box RNA helicase3 (DDX3) and have validated their oncogenic role in many cancers. Inhibition of COX-2 and DDX3 offers a potential pharmacological strategy for prevention of cancer progression. The COX-2 isoform is expressed in response to pro-inflammatory stimuli in premalignant lesions, including cervical tissues. This study elucidates the potential role of plant derived compound Forskolin (FSK) in plummeting the expression of COX-2 and DDX3 in cervical cancer. To establish this, the cervical cancer cells were treated with the FSK compound which induced a dose dependent significant inhibition of COX-2 and DDX3 expression. The FSK treatment also significantly induced apoptosis in cancer cells by modulating the expression of apoptotic markers like caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, full length-poly ADP ribose polymerase (PARP), cleaved-poly ADP ribose polymerase (C-PARP) and Bcl2 in dose dependent manner. Further FSK significantly modulated the cell survival pathway Phosphatidylinositol 3-kinase (PI3-K)/Akt signalling pathway upon 24 h of incubation in cervical cancer cells. The molecular docking studies revealed that the FSK engaged the active sites of both the targets by interacting with key residues.

Significant growth inhibition by a bispecific affibody targeting oncoprotein E7 in both HPV16 and 18 positive cervical cancer in vitro and in vivo.

The infection with HPV 16 and 18 high-risk types account for more than 80 % of cervical cancer incidence, but there is still no targeted agent against HPV for cervical cancer therapy. Our previous study constructed a bispecific affibody Z16-18 targeting HPV16 and 18 early antigen 7 (E7, responsible for the infected cell malignant transformation). In the present study, we prepared Z16-18 in prokaryotic expression system and confirmed its significant growth inhibition both on SiHa (HPV16 positive) and HeLa (HPV18 positive) cervical cancer cells by arresting cell cycle at G0/G1 phase. The IC50 of Z16-18 on SiHa and HeLa were close in value. Z16-18 could specifically target E7 in both SiHa and HeLa, and exhibited prominent targeted enrichment on tumor tissues derived from SiHa or HeLa, resulting in the inhibition of tumourigenesis and tumour growth in vivo. Furthermore, Z16-18 could inhibit the interaction between E7 and pRb to block the E7-pRb carcinogenic pathway, resulting in the decreased release of E2F and the cell growth inhibition characterized by the decrease of CDK6 and Cyclin D1. This study provides a new strategy for targeted therapy based on affibody, and Z16-18 has great potential for utilisation and development as an agent targeting HPV16 and HPV18 related cervical cancer.

Synergistic Cytotoxicity Effect of 5-Fluorouracil and SHP2 Inhibitor Demethylincisterol A3 on Cervical Cancer Cell.

BACKGROUND: Demethylincisterol A3 (DTA3) has been identified as an SHP2 inhibitor and suppresses the growth of many cancer cells. 5-Fluorouracil (5-FU) is widely used for the clinical treatment of various cancers. However, the combination effects of 5-FU and DTA3 on cervical cancer cells remain unknown. OBJECTIVE: This study evaluates the mechanism of the combination effects of 5-FU and DTA3 in cervical cancer cells. METHODS: The synergistic cytotoxic effects of 5-FU and DTA3 in cervical cancer cells were calculated. Apoptosis was analysed by flow cytometry. Western blot analyses were used to examine the related signalling pathways. RESULTS: DTA3 and 5-FU synergized to induce apoptosis and repress proliferation of cervical cancer cells by downregulating the activation of PI3K/AKT and NF-κB signalling pathways. We provided evidence that the upregulation of SHP2 expression by transfection significantly inhibited the cytotoxicity of 5-FU and DTA3. SHP2 knockdown enhanced the anti-proliferation activity of 5-FU, indicating targeting SHP2 sensitized cervical cancer cells to 5-FU. CONCLUSION: Our study demonstrates that SHP2 inhibitor DTA3 and 5-FU have a synergistic cytotoxic effect on cervical cancer cells. The synergistic combination of SHP2 inhibitor and 5-FU may present a promising strategy for the treatment of cervical cancer.

In vivo and in vitro studies of Alloimperatorin induced autophagy in cervical cancer cells via reactive oxygen species pathway.

Alloimperatorin (Alloi) has been shown to have anti-proliferative effects in our previous studies. we aimed to investigate whether Alloimperatorin induces autophagy through the reactive oxygen species (ROS) pathway and anticancer activity in vivo. The anti-proliferative effect of Alloimperatorin was evaluated using a cell counting kit (CCK-8 kit). Apoptosis was detected using flow cytometry. Confocal microscopy, immunofluorescence, and mRFP-GFP-LC3 lentivirus transfection were used to verify autophagy. Electron microscopy detection of autophagosomes was induced by Alloimperatorin. Western blotting was used to detect autophagy proteins in HeLa and SiHa cells. A xenograft model was used to monitor the inhibitory effect of Alloimperatorin on tumor growth in nude mice. The results showed that Alloimperatorin induced ROS production and inhibited the proliferation of HeLa and SiHa cells. Furthermore, Alloimperatorin increased the apoptosis rate in HeLa and SiHa cells. Confocal microscopy fluorescence indicated that Alloimperatorin increased autophagy fluorescence of HeLa and SiHa cells. mRFP-GFP-LC3 lentivirus transfection and electron microscopy demonstrated that Alloimperatorin increased autophagy in HeLa and SiHa cells. Western blotting showed that Alloimperatorin induced the expression of autophagy proteins in HeLa and SiHa cells. However, N-acetylcysteine reversed the autophagy. These results demonstrate that Alloimperatorin can induce autophagy in HeLa and SiHa cells through the ROS pathway. In vivo xenograft experiments showed that Alloimperatorin could inhibit tumor growth in nude mice. Alloimperatorin is expected to be an effective new drug for cervical cancer treatment.Abbreviations: ROS, reactive oxygen species; Alloi, Alloimperatorin; CCK-8, Cell Counting Kit-8; NAC, N-acetyl-L-cysteine; DCFH-DA, 2,7-dichlorodihydrofluorescein diacetate; OD, optical density; PBS, phosphate buffer solution; BCA, bicinchoninic acid; DAPI, 4,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide.

Phytochemical and antiplasmodial investigation of Gardenia thunbergia L. f. leaves.

Previous results indicated that the methanol extract of Gardenia thunbergia has antiplasmodial activity but no compounds have ever been isolated from the plant. Therefore, this study aimed to investigate the phytochemical and antiplasmodial properties of the plant. The methanol leaf extract of G. thunbergia inhibited Plasmodium falciparum at 50 µg/mL (> 80% inhibition) and was not cytotoxic against HeLa cells. Chromatographic purification of the extract afforded a new saponin and eight other known compounds. The saponin and two flavonoid glycosides displayed non-selective antiplasmodial activity at 50 µg/mL but the activities were diminished at 10 µg/mL. The presence of the isolated compounds in the leaf extract of G. thunbergia could account for the folkloric use of the plant in treating malaria.

Anticancer Potential of L-Histidine-Capped Silver Nanoparticles against Human Cervical Cancer Cells (SiHA).

This study reports the synthesis of silver nanoparticles using amino acid L-histidine as a reducing and capping agent as an eco-friendly approach. Fabricated L-histidine-capped silver nanoparticles (L-HAgNPs) were characterized by spectroscopic and microscopic studies. Spherical shaped L-HAgNPs were synthesized with a particle size of 47.43 ± 19.83 nm and zeta potential of -20.5 ± 0.95 mV. Results of the anticancer potential of L-HAgNPs showed antiproliferative effect against SiHa cells in a dose-dependent manner with an IC50 value of 18.25 ± 0.36 µg/mL. Fluorescent microscopic analysis revealed L-HAgNPs induced reactive oxygen species (ROS) mediated mitochondrial dysfunction, leading to activation of apoptotic pathway and DNA damage eventually causing cell death. To conclude, L-HAgNPs can act as promising candidates for cervical cancer therapy.